ABERRANT PROCESSING OF WILD-TYPE AND MUTANT BOVINE PANCREATIC TRYPSIN-INHIBITOR SECRETED BY ASPERGILLUS-NIGER

Bovine pancreatic trypsin inhibitor (BPTI) was secreted by Aspergillus niger at yields of up to 23 mg l(-1) using a protein fusion strategy. BPTI was linked to part of the fungal glucoamylase protein (GAM) with a dibasic amino acid (KEX2) processing site at the fusion junction. E lectrospray ionisation mass spectrometry and N-terminal protein sequen cing revealed that, although biologically active in vitro, the purifie d products from a number of independent transformants consisted of a m ixture of BPTI molecules differing at the N-terminus. Approximately 35 -60% of this mixture was processed correctly. Aberrant processing of t he GAM-BPTI fusion protein by the A. niger KEX2-like endoprotease was the most likely cause of this variation although the involvement of ot her fungal endoproteases could not be ruled out. In vitro studies have highlighted a weak interaction between BPTI and the Saccharomyces cer evisiae KEX2 endoprotease, suggesting that BPTI is not a potent inhibi tor of KEX2p. A small proportion of the recombinant BPTI (10%) showed 'nicking' of the K15-A16 bond, indicating an interaction with a fungal trypsin-like enzyme. Mutant BPTI homologues designed to have anti-ela stase activity, BPTI(K15V), BPTI(K15V,P13I) and BPTI(K15V,G12A), have also been expressed and secreted by A. niger. They also showed a simil ar spectrum of aberrant N-terminal processing but no 'nicking' of the K15-V16 bond was observed. Comparison of A. niger with other expressio n systems showed that it is an effective system for producing BPTI and its homologues, although not all molecules were correctly processed. This variation in processing efficiency may be useful in understanding the important determinants of protein processing in this fungus. (C) 1998 Elsevier Science B.V. All rights reserved.